Method for treating atherosclerosis by administering human MCP1 fusions

ABSTRACT

The present invention provides polypeptides including MCP1 fused, optionally, by a linker, to an immunoglobulin. Methods for using the polypeptides to treat medical disorders are also covered.

The present application is a continuation of U.S. patent applicationSer. No. 12/621,228; filed Nov. 18, 2009; which is a divisionalapplication of U.S. patent application Ser. No. 11/502,064; filed Aug.10, 2006; which claims the benefit of U.S. provisional patentapplication No. 60/707,731, filed Aug. 12, 2005, each of which areherein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to fusion proteins that are useful for,inter alia, treatment or prevention of inflammatory conditions; alongwith method for treating such conditions using the fusion proteins.

BACKGROUND OF THE INVENTION

Monocyte chemotactic protein-1 (MCP1, also known as HC11, MCAF, CCL2,SCYA2, GDCF-2, SMC-CF, HSMCR30, MGC9434, GDCF-2 and HC11) is a highlyselective, high-affinity chemokine ligand for chemokine receptor CCR2.It is secreted locally by inflammatory tissues to attract CCR2-bearingcells such as monocytes and memory T cells. Upon binding to CCR2, MCP1induces calcium flux and cell migration toward the gradient of MCP1.

There is substantial biological and genetic evidence for the criticalinvolvement of MCP1 and CCR2 in inflammatory diseases includingarthritis, atherosclerosis, multiple sclerosis, and fibrosis. Micedeficient in either MCP1 or CCR2 were protected from developingexperimental autoimmune encephalomyelitis (EAE) (Huang et al., 2001, J.Exp. Med. 193:713; Izikson et al., 2000, J. Exp. Med. 192:1075; Mahadand Ransohoff, 2003, Semi. Immunol. 15:23). CCR2-deficient mice werealso protected from collagen-induced arthritis (Quinones et al., 2004,J. Olin. Invest. 113:856). Furthermore, CCR2-knockout mice wereresistant to the development of atherosclerotic plaques (Charo andTaubman, 2004, Circ. Res. 95:858).

There exists a need in the art for anti-inflammatory agents that targetCCR2-mediated inflammation.

SUMMARY OF THE INVENTION

The invention disclosed herein stems from the surprising discovery thatthe targeting of a chemokine receptor by desensitizing the receptor withthe systemic administration of a chemokine ligand, leads to theprevention of the receptor-bearing cells from trafficking toinflammatory sites and from exposure to activation signals such asinterferon-gamma. In the exemplary examples discussed herein, CCR2 wasused as the chemokine receptor and MCP1 as the chemokine ligand. Toextend the serum half-life of the MCP1 chemokine animmunoglobulin-fusion protein was designed by tagging the MCP1 with theimmunoglobulin constant region (from the hinge to the CH3 regions).

The present invention addresses the need for anti-inflammatory therapiesand other needs by providing, in an embodiment of the invention, achemokine ligand, such as MCP1, SDF1 or MIP1β or a fragment thereof(e.g., mature polypeptide thereof), fused to an agent that extends thein vivo half-life of the protein, for example, an immunoglobulin or afragment thereof or PEG.

The present invention provides an isolated polypeptide comprising (1)one or more chemokine polypeptides fused to one or more half-lifeextending moieties; or (2) two or more fused chemokine polypeptides. Inan embodiment of the invention, the MCP1 is a member selected from thegroup consisting of human MCP1 and mouse MCP1. In an embodiment of theinvention, the moiety is polyethylene glycol (PEG) or an immunoglobulin.In an embodiment of the invention, the polypeptide comprises one or moremature MCP1 polypeptides (e.g., human or mouse) fused to one or moreimmunoglobulins (e.g., any of SEQ ID NOs: 8-12). In an embodiment of theinvention, the MCP1 is a member selected from the group consisting ofhuman MCP1 and mouse MCP1. In an embodiment of the invention, theimmunoglobulin is a member selected from the group consisting of γ1 andγ4 from the hinge to the CH3 region. In an embodiment of the invention,the polypeptide is a member selected from the group consisting of:mature human MCP1 fused to mouse IgG1; mature human MCP1 fused to humanIgG4; mature human MCP1 fused to human IgG4 monomeric variant; maturehuman MCP1 fused to human IgG1; and mature human MCP1 fused to humanIgG1 monomeric variant. In an embodiment of the invention, the MCP1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2. In an embodiment of the invention, the immunoglobulincomprises an amino acid sequence set forth in SEQ ID NOs: 3-7. In anembodiment of the invention, the MCP1 is fused to the immunoglobulin bya peptide linker (e.g., GS). The scope of the present invention includesa pharmaceutical composition comprising any of the polypeptides and apharmaceutically acceptable carrier. Also within the scope of thepresent invention is any of the polypeptides herein in association withone or more further therapeutic agents or a pharmaceutical compositionthereof; for example, wherein the further therapeutic agent is a memberselected from the group consisting of aspirin, diclofenac, diflunisal,etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid,meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam,salsalate, sulindac, tenoxicam, tiaprofenic acid, tolmetin,betamethasone benzoate, betamethasone valerate, clobetasol propionate,desoximetasone, fluocinolone acetonide, flurandrenolide, a topicalsteroid, alclometasone dipropionate, aloe vera, amcinonide, amcinonide,anthralin, betamethasone dipropionate, betamethasone valerate,calcipotriene, clobetasol propionate, coal tar, Dead Sea salts,desonide, desonide; betamethasone valerate, desoximetasone, diflorasonediacetate, epsom salts, fluocinolone acetonide, fluocinonide,flurandrenolide, fluticasone propionate, halcinonide, halobetasolpropionate, hydrocortisone valerate, hydrocortisone, mometasone furoate,oilated oatmeal, petroleum jelly, prednicarbate, salicylic acid,tazarotene, triamcinolone acetonide, a mixture of hydrocortisone,dexamethasone, methylprednisolone and prednisolone, alefacept,etanercept, cyclosporine, methotrexate, acitretin, isotretinoin,hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-Thioguanine,anakinra, injectable gold, penicillamine, azathioprine, chloroquine,hydroxychloroquine, sulfasalazine, oral gold, auranofin, gold sodiumthiomalate, aurothioglucose, mesalamine, sulfasalazine, budesonide,metronidazole, ciprofloxacin, azathioprine, 6-mercaptopurine or dietarysupplementation of calcium, folate, vitamin B12, celecoxib, rofecoxib,valdecoxib, lumiracoxib, etoricoxib, efalizumab, adalimumab, infliximaband ABX-IL8.

The present invention provides an isolated polynucleotide encoding anyof the polypeptides herein—e.g., encoding a member selected from thegroup consisting of human unprocessed MCP1 fused to mouse IgG1; humanunprocessed MCP1 fused to human IgG4; human unprocessed MCP1 fused tohuman IgG4 monomeric variant; human unprocessed MCP1 fused to humanIgG1; and human unprocessed MCP1 fused to human IgG1 monomeric variant.In an embodiment of the invention, the MCP1 is encoded by a nucleotidesequence set forth in SEQ ID NO: 13. In an embodiment of the invention,the immunoglobulin is encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 14-18. In an embodiment of theinvention, the nucleotide sequence is selected from the group consistingof SEQ ID NOs: 19-23. The present invention also includes an isolatedvector (e.g., as shown in FIG. 1 and/or comprising the nucleotidesequence of SEQ ID NO: 24) comprising the polynucleotide (e.g.,pcDNA3.1(+)hMCP1 mIgG) as well as a host cell comprising the vector.

The present invention also provides a method for making an MCP1-Igpolypeptide comprising transforming a host cell with an expressionvector (e.g., pcDNA3.1(+)hMCP1 mIgG) comprising a polynucleotideencoding said polypeptide under conditions suitable for said expressionand, optionally, isolating the polypeptide from the host cell. In anembodiment of the invention, the polynucleotide encodes a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 8-12. Any polypeptide made by the method is also within thescope of the present invention.

The present invention provides a method for treating a disordertreatable by decreasing expression or activity of a chemokine ligand(e.g., MCP1, SDF1 or MIP1β) or chemokine receptor or by decreasing themigration of chemokine receptor bearing cells into inflammatory tissues,in a subject, comprising desensitizing chemokine receptor bearing cells,in the subject, to chemokine ligand. In an embodiment of the invention,the disorder is a member selected from the group consisting of aninflammatory medical disorder, parasitic infection, bacterial infection,viral infection, cancer, a cardiovascular disorder and a circulatorydisorder. In an embodiment of the invention, the chemokine receptorbearing cells are desensitized to the chemokine ligand by systemicadministration of chemokine ligand polypeptide or a chemokine ligandpolypeptide fused to a half-life extending moiety (e.g., immunoglobulinor fragment thereof). In an embodiment of the invention, the MCP1comprises an amino acid sequence set forth in SEQ ID NO: 2. In anembodiment of the invention, the Ig comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 3-7. In an embodimentof the invention, the polypeptide is MCP1-Ig comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 8-12.

The present invention provides a method for treating or preventing aninflammatory medical disorder in a subject comprising administering, tothe subject, MCP1-Ig or a pharmaceutical composition thereof optionallyin association with a further therapeutic agent or procedure. In anembodiment of the invention, the disorder is a member selected from thegroup consisting of appendicitis, peptic ulcer, gastric ulcer andduodenal ulcer, peritonitis, pancreatitis, inflammatory bowel disease,colitis, ulcerative colitis, pseudomembranous colitis, acute colitis,ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis,cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis,Whipple's disease, asthma, allergy, anaphylactic shock, immune complexdisease, organ ischemia, reperfusion injury, organ necrosis, hay fever,sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia,eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion,epididymitis, vaginitis, prostatitis, and urethritis, bronchitis,emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratorydistress syndrome, pneumoultramicroscopicsilicovolcanoconiosis,alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, dermatitis,atopic dermatitis, dermatomyositis, sunburn, urticaria warts, wheals,stenosis, restenosis, vasulitis, angiitis, endocarditis, arteritis,atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardialischemia, periarteritis nodosa, rheumatic fever, meningitis,encephalitis, multiple sclerosis, neuritis, neuralgia, uveitis,arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease,gout, periodontal disease, rheumatoid arthritis, synovitis, myastheniagravis, thryoiditis, systemic lupus erythematosus, Goodpasture'ssyndrome, Behcets's syndrome, allograft rejection and graft-versus-hostdisease. In an embodiment of the present invention, the MCP1 comprisesan amino acid sequence is set forth in SEQ ID NO: 2. In an embodiment ofthe present invention, the Ig comprises amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 3-7. In an embodiment of thepresent invention, the MCP1-Ig comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8-12. In an embodiment of thepresent invention, the subject is a human. In an embodiment of thepresent invention, the further therapeutic agent or procedure is amember selected from the group consisting of aspirin, diclofenac,diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid,meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam,salsalate, sulindac, tenoxicam, tiaprofenic acid, tolmetin,betamethasone benzoate, betamethasone valerate, clobetasol propionate,desoximetasone, fluocinolone acetonide, flurandrenolide, a topicalsteroid, alclometasone dipropionate, aloe vera, amcinonide, amcinonide,anthralin, betamethasone dipropionate, betamethasone valerate,calcipotriene, clobetasol propionate, coal tar, Dead Sea salts,desonide, desonide; betamethasone valerate, desoximetasone, diflorasonediacetate, epsom salts, fluocinolone acetonide, fluocinonide,flurandrenolide, fluticasone propionate, halcinonide, halobetasolpropionate, hydrocortisone valerate, hydrocortisone, mometasone furoate,oilated oatmeal, petroleum jelly, prednicarbate, salicylic acid,tazarotene, triamcinolone acetonide, a mixture of hydrocortisone,dexamethasone, methylprednisolone and prednisolone, alefacept,etanercept, cyclosporine, methotrexate, acitretin, isotretinoin,hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-Thioguanine,anakinra, injectable gold, penicillamine, azathioprine, chloroquine,hydroxychloroquine, sulfasalazine, oral gold, auranofin, gold sodiumthiomalate, aurothioglucose, mesalamine, sulfasalazine, budesonide,metronidazole, ciprofloxacin, azathioprine, 6-mercaptopurine or dietarysupplementation of calcium, folate, vitamin B12, celecoxib, rofecoxib,valdecoxib, lumiracoxib, etoricoxib, efalizumab, adalimumab, infliximab,ABX-IL8 and phototherapy.

The present invention provides a method for increasing the in vivohalf-life of MCP1 in the body of a subject comprising fusing MCP1 to animmunoglobulin or a fragment thereof. In an embodiment of the presentinvention, the MCP1 comprises an amino acid sequence is set forth in SEQID NO: 2. In an embodiment of the present invention, the immunoglobulincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 3-7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Map of plasmid pcDNA3.1(+)hMCP1 mIgG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a polypeptide comprising MCP1 fused toany half-life extending moiety that extends or prolongs the in vivohalf-life of MCP1 in the body (e.g., in the plasma) of a subject.

Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel, et al.,(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” includesthe phosphate ester polymeric form of ribonucleosides (adenosine,guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester analogs thereof, such asphosphorothioates and thioesters, in single stranded form,double-stranded form or otherwise.

A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotidesequence” is a series of nucleotide bases (also called “nucleotides”) ina nucleic acid, such as DNA or RNA, and means any chain of two or morenucleotides.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in production of the product.

The term “gene” means a DNA sequence that codes for or corresponds to aparticular sequence of ribonucleotides or amino acids which comprise allor part of one or more RNA molecules, proteins or enzymes, and may ormay not include regulatory DNA sequences, such as promoter sequences,which determine, for example, the conditions under which the gene isexpressed. Genes may be transcribed from DNA to RNA which may or may notbe translated into an amino acid sequence.

A “protein sequence”, “peptide sequence” or “polypeptide sequence” or“amino acid sequence” includes a series of two or more amino acids in aprotein, peptide or polypeptide.

The terms “isolated polynucleotide” or “isolated polypeptide” include apolynucleotide (e.g., RNA or DNA molecule, or a mixed polymer) or apolypeptide, respectively, which are partially or fully separated fromother components that are normally found in cells or in recombinant DNAexpression systems. These components include, but are not limited to,cell membranes, cell walls, ribosomes, polymerases, serum components andextraneous genomic sequences.

An isolated polynucleotide or polypeptide will, preferably, be anessentially homogeneous composition of molecules but may contain someheterogeneity.

“Amplification” of DNA as used herein includes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki, et al., Science (1988) 239:487.

The term “host cell” includes any cell of any organism that is selected,modified, transfected, transformed, grown, or used or manipulated in anyway, for the production of a substance by the cell, for example theexpression or replication, by the cell, of a gene, a DNA or RNA sequenceor a protein. Host cells include bacterial cells (e.g., E. coli),Chinese hamster ovary (CHO) cells, HEK293 cells, myeloma cells includingbut not limited to SP2/0, NS1, and NS0, murine macrophage J774 cells orany other macrophage cell line and human intestinal epithelial Caco2cells.

The nucleotide sequence of a polynucleotide may be determined by anymethod known in the art (e.g., chemical sequencing or enzymaticsequencing). “Chemical sequencing” of DNA includes methods such as thatof Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), inwhich DNA is randomly cleaved using individual base-specific reactions.“Enzymatic sequencing” of DNA includes methods such as that of Sanger(Sanger, et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).

The polynucleotides herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like.

In general, a “promoter” or “promoter sequence” is a DNA regulatoryregion capable of binding an RNA polymerase in a cell (e.g., directly orthrough other promoter-bound proteins or substances) and initiatingtranscription of a coding sequence. A promoter sequence is, in general,bounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at any level. Within thepromoter sequence may be found a transcription initiation site(conveniently defined, for example, by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. The promoter may be operably associatedwith other expression control sequences, including enhancer andrepressor sequences or with a polynucleotide of the invention. Promoterswhich may be used to control gene expression include, but are notlimited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and5,168,062), the SV40 early promoter region (Benoist, et al., (1981)Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797),the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl.Acad. Sci. USA 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster, et al., (1982) Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl.Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American (1980) 242:74-94; and promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or thealkaline phosphatase promoter.

A coding sequence is “under the control of”, “functionally associatedwith” or “operably associated with” transcriptional and translationalcontrol sequences in a cell when the sequences direct RNA polymerasemediated transcription of the coding sequence into RNA, preferably mRNA,which then may be RNA spliced (if it contains introns) and, optionally,translated into a protein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene, RNA or DNA sequence to become manifest; forexample, producing a protein by activating the cellular functionsinvolved in transcription and translation of a corresponding gene. A DNAsequence is expressed in or by a cell to form an “expression product”such as an RNA (e.g., mRNA) or a protein. The expression product itselfmay also be said to be “expressed” by the cell.

The term “transformation” means the introduction of polynucleotide intoa cell. The introduced gene or sequence may be called a “clone”. A hostcell that receives the introduced DNA or RNA has been “transformed” andis a “transformant” or a “clone.” The DNA or RNA introduced to a hostcell can come from any source, including cells of the same genus orspecies as the host cell, or from cells of a different genus or species.

The term “vector” includes a vehicle (e.g., a plasmid) by which a DNA orRNA sequence can be introduced into a host cell, so as to transform thehost and, optionally, promote expression and/or replication of theintroduced sequence.

Vectors that can be used in this invention include plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles that mayfacilitate introduction of the polynucleotide into the host. Plasmidsare the most commonly used form of vector but all other forms of vectorswhich serve a similar function and which are, or become, known in theart are suitable for use herein. See, e.g., Pouwels, et al., CloningVectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N.Y., andRodriguez et al. (eds.), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, 1988, Buttersworth, Boston, Mass.

The present invention comprises a method for producing MCP1 (e.g., amature MCP1 polypeptide), an MCP1 multimer or a fusion thereof (e.g.,fused to an in vivo half-life extending moiety (e.g., Ig)) (e.g., in anexpression system). In an embodiment of the invention, the MCP1 ormultimer or fusion thereof is inserted into an expression vector whichis introduced into a suitable host cell. The protein is then allowed toexpress in the host cell. The protein may then be isolated from the hostcell and purified further. Any polypeptide produced by such a method isalso within the scope of the present invention.

The term “expression system” means a host cell and compatible vectorwhich, under suitable conditions, can express a protein or nucleic acidwhich is carried by the vector and introduced to the host cell. Commonexpression systems include E. coli host cells or mammalian host cells(e.g., CHO cells, HEK293 cells, and myeloma cells) and plasmid vectors,insect host cells and Baculovirus vectors, and mammalian host cells andvectors.

Expression of nucleic acids encoding MCP1 or multimer or fusion thereofof this invention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although E. coli host cells areemployed most frequently in prokaryotic systems, many other bacteria,such as various strains of Pseudomonas and Bacillus, are known in theart and can be used as well. Suitable host cells for expressing nucleicacids encoding the polypeptides include prokaryotes and highereukaryotes. Prokaryotes include both gram-negative and gram-positiveorganisms, e.g., E. coli and B. subtilis. Higher eukaryotes includeestablished tissue culture cell lines from animal cells (e.g., CHOcells), both of non-mammalian origin, e.g., insect cells, and birds, andof mammalian origin, e.g., human, primates, and rodents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. A representative vector for amplifying DNA ispBR322 or any of many of its derivatives (e.g., pUC18 or 19). Vectorsthat can be used to express the MCP1 polypeptides include, but are notlimited to, those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al.,“Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derivedPromoters”, in Rodriguez and Denhardt (eds.) Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston,pp. 205-236. Many polypeptides can be expressed, at high levels, in anE. coli/T7 expression system as disclosed in U.S. Pat. Nos. 4,952,496,5,693,489 and 5,869,320 and in Davanloo, P., et al., (1984) Proc. Natl.Acad. Sci. USA 81: 2035-2039; Studier, F. W., et al., (1986) J. Mol.Biol. 189: 113-130; Rosenberg, A. H., et al., (1987) Gene 56: 125-135;and Dunn, J. J., et al., (1988) Gene 68: 259.

The T7 expression system comprises pET plasmids which contain anexpression cassette in which the gene of interest (e.g., MCP1 ormultimer or fusion thereof) is inserted behind an extremely strongpromoter from the E. coli bacteriophage T7 (Studier et al., J. Mol.Biol. 189(1):113-30 (1986)). In the absence of the T7 polymerase, thispromoter is shut off. For expression to occur, the pET plasmids aretransformed into bacteria strains that typically contain a single copyof the T7 polymerase on the chromosome in a lambda lysogen (the mostcommonly used lysogen is known as DE3). The T7 polymerase is under thecontrol of the Lac-UV5 lac promoter. When cells are grown in mediawithout lactose, the lac repressor (lacI) binds to the lac operator andprevents transcription from the lac promoter. When lactose is the solecarbon source, or when the lactose analogisopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the media,lactose (or IPTG) binds to the repressor and induces its dissociationfrom the operator, permitting transcription from the promoter. Additionof glucose to the culture media contributes to repression of the T7 RNApolymerase via the mechanism of catabolite repression.

Higher eukaryotic tissue culture cells may also be used for therecombinant production of the MCP1 or multimer or fusion thereof of theinvention. Higher eukaryotic tissue culture cell line can be used,including insect baculovirus expression systems and mammalian cells.Transformation or transfection and propagation of such cells have becomea routine procedure. Examples of useful cell lines include HeLa cells,Chinese hamster ovary (CHO) cell lines, HEK 293 cells, myeloma cells,J774 cells, Caco2 cells, baby rat kidney (BRK) cell lines, insect celllines, bird cell lines, and monkey (COS) cell lines. Typically,expression vectors for such cell lines include an origin of replication,a promoter, a translation initiation site, RNA splice sites (if genomicDNA is used), a polyadenylation site, and a transcription terminationsite. These vectors also, usually, contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Examples of expression vectors include pCR®3.1, pcDNA1,pCD (Okayama, et al., (1985) Mol. Cell. Biol. 5:1136), pMC1neo Poly-A(Thomas, et al., (1987) Cell 51:503), pREP8, pSVSPORT and derivativesthereof, and baculovirus vectors such as pAC373 or pAC610.

In an embodiment of the invention, the MCP1-Ig can be purified byprotein A or protein G chromatography. Protein A and protein G bindpreferentially to immunoglobulins. Furthermore, the polypeptide can bepurified by standard methods, including, but not limited to, salt oralcohol precipitation, affinity chromatography, preparative disc-gelelectrophoresis, isoelectric focusing, high pressure liquidchromatography (HPLC), reversed-phase HPLC, gel filtration, cation andanion exchange and partition chromatography, and countercurrentdistribution. Such purification methods are well known in the art andare disclosed, e.g., in “Guide to Protein Purification”, Methods inEnzymology, Vol. 182, M. Deutscher, Ed., 1990, Academic Press, New York,N.Y. Where a polypeptide is being isolated from a cellular or tissuesource, it is preferable to include one or more inhibitors ofproteolytic enzymes in the assay system, such as phenylmethanesulfonylfluoride (PMSF), Pefabloc SC, pepstatin, leupeptin, chymostatin andEDTA.

Modifications (e.g., post-translational modifications) that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications, in large part, will be determined by thehost cell's post-translational modification capacity and themodification signals present in the polypeptide amino acid sequence. Forinstance, as is well known, glycosylation often does not occur inbacterial hosts such as E. coli. Accordingly, when glycosylation of MCP1or the multimer or fusion thereof is desired, the polypeptide can beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out post-translational glycosylations which aresimilar to those of mammalian cells. For this reason, insect cellexpression systems have been developed to express, efficiently,mammalian proteins having native patterns of glycosylation. An insectcell, which may be used in this invention, is any cell derived from anorganism of the class Insecta; for example, where the insect isSpodoptera fruigiperda (Sf9 or Sf21) or Trichoplusia ni (High 5).Examples of insect expression systems that can be used with the presentinvention, for example to produce MCP1 or a multimer or fusion thereof,include Bac-To-Bac (Invitrogen Corporation, Carlsbad, Calif.) or Gateway(Invitrogen Corporation, Carlsbad, Calif.). If desired, deglycosylationenzymes can be used to remove carbohydrates attached during productionin eukaryotic expression systems.

Pegylation or addition of polyethylene glycol (PEG) to a polypeptidesuch as MCP1 or a fusion or multimer thereof can be accomplished usingconventional and well known methods in the art (see e.g., U.S. Pat. No.5,691,154; U.S. Pat. No. 5,686,071; U.S. Pat. No. 5,639,633; U.S. Pat.No. 5,492,821; U.S. Pat. No. 5,447,722; U.S. Pat. No. 5,091,176).

The present invention contemplates any superficial or slightmodification to the amino acid or nucleotide sequences which correspondto the MCP1 or a multimer or fusion thereof of the invention. Inparticular, the present invention contemplates sequence conservativevariants of the polynucleotides which encode the polypeptides of theinvention. “Sequence-conservative variants” of a polynucleotide sequenceare those in which a change of one or more nucleotides in a given codonresults in no alteration in the amino acid encoded at that position.Function-conservative variants of the polypeptides of the invention arealso contemplated by the present invention. “Function-conservativevariants” are those in which one or more amino acid residues in aprotein or enzyme have been changed without altering the overallconformation and function of the polypeptide, including, but, by nomeans, limited to, replacement of an amino acid with one having similarproperties. Amino acids with similar properties are well known in theart. For example, polar/hydrophilic amino acids which may beinterchangeable include asparagine, glutamine, serine, cysteine,threonine, lysine, arginine, histidine, aspartic acid and glutamic acid;nonpolar/hydrophobic amino acids which may be interchangeable includeglycine, alanine, valine, leucine, isoleucine, proline, tyrosine,phenylalanine, tryptophan and methionine; acidic amino acids which maybe interchangeable include aspartic acid and glutamic acid and basicamino acids which may be interchangeable include histidine, lysine andarginine.

The present invention includes polynucleotides encoding an MCP1 (e.g., amature MCP1 polypeptide), an MCP1 multimer or a fusion thereof (e.g.,fused to an in vivo half-life extending moiety (e.g., Ig)) (e.g., any ofSEQ ID NOs: 1, 2, 8-12) as well as nucleic acids which hybridize to thepolynucleotides. Preferably, the nucleic acids hybridize under lowstringency conditions, more preferably under moderate stringencyconditions and most preferably under high stringency conditions. Anucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook, et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. Typical low stringency hybridization conditions are 55°C., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide at 42° C.; or 30%formamide, 5×SSC, 0.5% SDS at 42° C. Typical, moderate stringencyhybridization conditions are similar to the low stringency conditionsexcept the hybridization is carried out in 40% formamide, with 5× or6×SSC at 42° C. High stringency hybridization conditions are similar tolow stringency conditions except the hybridization conditions arecarried out in 50% formamide, 5× or 6×SSC and, optionally, at a highertemperature (e.g., higher than 42° C.: 57° C., 59° C., 60° C., 62° C.,63° C., 65° C. or 68° C.). In general, SSC is 0.15M NaCl and 0.015MNa-citrate. Hybridization requires that the two nucleic acids containcomplementary sequences, although, depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the higher the stringency under which the nucleicacids may hybridize. For hybrids of greater than 100 nucleotides inlength, equations for calculating the melting temperature have beenderived (see Sambrook, et al., supra, 9.50-9.51). For hybridization withshorter nucleic acids, i.e., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook, et al., supra).

Also included in the present invention are polynucleotides comprisingnucleotide sequences and polypeptides comprising amino acid sequenceswhich are at least about 70% identical, preferably at least about 80%identical, more preferably at least about 90% identical and mostpreferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%,100%) to the reference MCP1 (e.g., a mature MCP1 polypeptide), MCP1multimer or fusion thereof (e.g., fused to an in vivo half-lifeextending moiety (e.g., Ig)) nucleotide sequence of any of SEQ ID NOs:13 and 19-23 and amino acid sequence of any of SEQ ID NOs: 1, 2, and8-12 when the comparison is performed by a BLAST algorithm wherein theparameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences.

Polypeptides comprising amino acid sequences which are at least about70% similar, preferably at least about 80% similar, more preferably atleast about 90% similar and most preferably at least about 95% similar(e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference MCP1 (e.g., amature MCP1 polypeptide), MCP1 multimer or fusion thereof (e.g., fusedto an in vivo half-life extending moiety (e.g., Ig)) of any of SEQ IDNOs: 1, 2 and 8-12, when the comparison is performed with a BLASTalgorithm wherein the parameters of the algorithm are selected to givethe largest match between the respective sequences over the entirelength of the respective reference sequences, are also included in thepresent invention.

Sequence identity refers to exact matches between the nucleotides oramino acids of two sequences which are being compared. Sequencesimilarity refers to both exact matches between the amino acids of twopolypeptides which are being compared in addition to matches betweennonidentical, biochemically related amino acids. Biochemically relatedamino acids which share similar properties and may be interchangeableare discussed above.

The following references regarding the BLAST algorithm are hereinincorporated by reference: BLAST ALGORITHMS: Altschul, S. F., et al.,(1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet.3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141;Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang,J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993)Comput. Chem. 17:149-163; Hancock, J. M., et al., (1994) Comput. Appl.Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “Amodel of evolutionary change in proteins.” in Atlas of Protein Sequenceand Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp.345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M.,et al., “Matrices for detecting distant relationships.” in Atlas ofProtein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., etal., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl.Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol.Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

Chemokine Ligands and Fusions Thereof

The present invention comprises any fusion polypeptide comprising one ormore of any chemokine such as MCP1 polypeptide, fused, optionally by alinker peptide (e.g., GS), to one or more “half-life extendingmoieties”. The invention further comprises any chemokine ligand multimercomprising two or more chemokine polypeptides, or fragments thereoffused into a single continuous polypeptide chain.

In an embodiment of the invention, the chemokine polypeptide is MCP1,SDF1 (including SDF1α or SDF1β; see Genbank accession no. P48061) orMIP1β (see Genbank accession no. NP_(—)002975.1; AAA36656.1; AAA36752.1;AAA51576.1; AAA57256.1; AAB00790.1; AAX07292.1; CAA34291.1; CAA37722.2;CAA37723.1; or CAG46916.1). In an embodiment of the invention, thechemokine polypeptide is any member of the CCL or CXCL class ofchemokine, for example, any of CCL1, 2, 3, 4, 5, 6, 7, 8, 9/10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28; or anyof CXCL 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Forexample, in an embodiment of the invention, the chemokine is any memberselected from the group consisting of I309, MIP1α, MIP1β, RANTES, C10,MCP2, MCP3, CCF18, eotaxin1, MCP4, MCP5, HCC1, HCC2, NCC4, TARC, PARK,ELC, LARC, SLC, MDC, MPIF1, eotaxin2, TECK, eotaxin3, ALP, CTACK. Forexample, in an embodiment of the invention, the chemokine is any memberselected from the group consisting of CCL23, CCL28, GROα, GROβ GROγ,PF4, ENA78, GCP2, PBP, β-TG, CTAP-III, NAP-2, IL-8, MIG, IP10, I-TAC,SDF1, BLC, BRAK, lungine, lymphotactin or fractalkine. The presentinvention comprises fusions comprising more than 1 chemokine fused to ahalf-life extending moiety (e.g., MCP1-SDF1-Ig).

A “half-life extending moiety” is any moiety, for example, apolypeptide, small molecule or polymer, that, when appended topolypeptide, extends the in vivo half-life of that polypeptide in thebody of a subject (e.g., in the plasma of the subject). For example, ahalf-life extending moiety is, in an embodiment of the invention,polyethylene glycol (PEG), monomethoxy PEG (mPEG) or an immunoglobulin(Ig). In an embodiment of the invention, PEG is a 5, 10, 12, 20, 30, 40or 50 kDa moiety or larger or comprises about 12000 ethylene glycolunits (PEG12000).

The term “Ig” or “immunoglobulin” includes any immunoglobulin from anyspecies, for example from human or from mouse as well as any fragment orvariant or mutant thereof. The term includes any heavy chain IgG, forexample, IgG1, IgG2, IgG3 or IgG4; IgA, for example, IgA1 or IgA2; IgM,IgD and IgE. In an embodiment of the invention, “Ig” or “immunoglobulin”refers to a polypeptide derived from any of the foregoing from the hingeto the CH3 of the heavy chain. In an embodiment of the invention, “Ig”is a “monomeric variant” or “monomer” of immunoglobulin. A monomericvariant of an immunoglobulin does not dimerize with anotherimmunoglobulin (see e.g., SEQ ID NOs: 5 and 7). A monmeric variant ofany immunoglobulin can be constructed by mutation of one or moreresidues (e.g., cysteine residues) involved in immunoglobulindimerization. For example, the cysteine residues can be mutated to aserine.

The present invention also provides MCP1 multimers (e.g., (mature orunprocessed MCP1)_(n) wherein n=2, 3, 4, 5, 6, 7, 8, 9 or 10 or more).An MCP1 multimer comprises one or more MCP1 polypeptides or maturepolypeptides thereof fused to one or more other MCP1 polypeptides ormature polypeptides thereof.

The term “MCP1” includes any MCP1 gene or protein from any organism(e.g., from any mammal for example human, Pan troglodytes; Canisfamiliaris (see e.g., accession no. P52203); Gallus gallus; Bos Taurus(see e.g., accession no. P28291); Rattus norvegicus (see e.g., accessionno. XP_(—)213425); or Mus musculus) or any homologue or fragment thereof(e.g., mature MCP1). MCP1 is described by several synonyms includingCCL2, HC11, MCAF, MCP1, MCP1, SCYA2, GDCF-2, SMC-CF, HSMCR30, MGC9434,GDCF-2 and HC11. A mature MCP1 polypeptide lacks the leader sequencethat is present in the unprocessed or immature MCP1 polypeptide. Theleader sequence can be easily identified by a practitioner of ordinaryskill in the art. In an embodiment of the present invention, the MCP1leader sequence is amino acids 1-23 of SEQ ID NO: 1.

The term “MCP1-Ig” includes any polypeptide comprising one or more MCP1polypeptides (e.g., human or mouse) or fragments thereof fused, in anyway and in any orientation, to one or more immunoglobulin polypeptidesor fragments thereof (e.g., human IgG4 or IgG1 or a fragment thereofincluding only the hinge to CH3 region).

In an embodiment of the invention, an unprocessed polypeptide sequenceof human MCP1 comprises the following amino acid sequence:

(SEQ ID NO: 1) MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT.

In an embodiment of the invention, a mature polypeptide sequence ofhuman MCP1 comprises the following amino acid sequence:

(SEQ ID NO: 2) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT.

In an embodiment of the invention, mature MCP1 is amino acids 30-99 ofSEQ ID NO: 1. In an embodiment of the invention, mature MCP1 comprisesthe amino acids sequence of SEQ ID NO: 2 wherein a pyroglutamic acid isadded to the N-terminus and in another embodiment of the invention, themature MCP1 comprises the amino acid sequence of SEQ ID NO: 2 whereinthe N-terminal glutamine (Q) is replaced with pyroglutamic acid.

In an embodiment of the invention, mouse MCP1 comprises the amino acidsequence disclosed under UniProtKB/Swiss-Prot accession no. P10148 orunder accession no. NP_(—)035463 or IPI00108087.1. Embodiments of theinvention also include mature, processed versions of these polypeptides(e.g., wherein signal peptide amino acids 1-23 are removed). In anembodiment of the invention, the mouse MCP1 comprises the amino acidsequence:

(SEQ ID NO: 30) mqvpvmllgl lftvagwsih vlaqpdavna pltccysftskmipmsrles ykritssrcp keavvfvtkl krevcadpkkewvqtyiknl drnqmrsept tlfktasalr ssaplnvkltrkseanastt fstttsstsv gvtsvtvn

In an embodiment of the invention, a mature polypeptide sequence ofmouse immunoglobulin heavy chain constant region (hinge to CH3 only),isotype γ1 comprises the amino acid sequence:

(SEQ ID NO: 3) VPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

In an embodiment of the invention, a mature polypeptide sequence ofhuman immunoglobulin heavy chain constant region (hinge to CH3 only),isotype γ4 comprises the amino acid sequence:

(SEQ ID NO: 4) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In an embodiment of the invention, a mature polypeptide sequence ofhuman immunoglobulin heavy chain constant region (hinge to CH3 only),isotype γ4 monomeric variant (C to S mutations in the hinge underscored)comprises the amino acid sequence:

(SEQ ID NO: 5) ESKYGPP S PS S PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In an embodiment of the invention, a mature polypeptide sequence ofhuman immunoglobulin heavy chain constant region (hinge to CH3 only),isotype γ1 comprises the amino acid sequence:

(SEQ ID NO: 6) VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In an embodiment of the invention, a mature polypeptide sequence ofhuman immunoglobulin heavy chain constant region (hinge to CH3 only),isotype γ1 monomeric variant (C to S mutations in the hinge underscored)

(SEQ ID NO: 7) VEPKS S DKTHT S PP S PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In an embodiment of the invention, a polypeptide sequence of maturehuman MCP1 fused to mouse IgG1 (linker underscored) comprises the aminoacid sequence:

(SEQ ID NO: 8) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKTGSVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNH HTEKSLSHSPGK

In an embodiment of the invention, a polypeptide sequence of maturehuman MCP1 fused to human IgG4 (linker underscored) comprises the aminoacid sequence:

(SEQ ID NO: 9) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKTGSESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK

In an embodiment of the invention, a polypeptide sequence of maturehuman MCP1 fused to human IgG4 monomeric variant (linker underscored)comprises the amino acid sequence:

(SEQ ID NO: 10) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKTGSESKYGPPSPSSPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK

In an embodiment of the invention, a polypeptide sequence of maturehuman MCP1 fused to human IgG1 (linker underscored) comprises the aminoacid sequence:

(SEQ ID NO: 11) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKTGSVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In an embodiment of the invention, a polypeptide sequence of human MCP1fused to human IgG1 monomeric variant (linker underscored) comprises theamino acid sequence:

(SEQ ID NO: 12) QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKTGSVEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In an embodiment of the invention, a DNA sequence of human MCP1 codingregion comprises the nucleotide sequence (the initiation and the stopcodons in underscored; codon of the first amino acid of the maturepolypeptide in bold font):

(SEQ ID NO: 13) atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaac ccaaactccgaagacttga

In an embodiment of the invention, a DNA sequence of mouse heavy-chainimmunoglobulin constant region, γ1 isotype, starting from the aminoterminus of the hinge region and ending at the carboxy-terminus of theCH3 region (stop codon in underscored) comprises the nucleotidesequence:

(SEQ ID NO: 14) gtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacaaaaccccgggaggagcagttcaacagcactttccgttcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatga

In an embodiment of the invention, a DNA sequence of human heavy-chainimmunoglobulin constant region, γ4 isotype, starting from the aminoterminus of the hinge region and ending at the carboxy-terminus of theCH3 region (stop codon underscored) comprises the nucleotide sequence:

(SEQ ID NO: 15) gagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaag agcctctccctgtctctgggtaaatga

In an embodiment of the invention, a DNA sequence of human heavy-chainimmunoglobulin constant region, γ4 isotype, monomeric variant, startingfrom the amino terminus of the hinge region and ending at thecarboxy-terminus of the CH3 region (Cys to Ser changes markedunderscored; stop codon in bold font) comprises the nucleotide sequence:

(SEQ ID NO: 16)gagtccaaatatggtcccccatctccatcatctccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga

The codon coding for serine, in any immunoglobulin monomeric variant setforth herein wherein one or more cysteines have been mutated, can be anycodon that encodes the serine amino acid; for example, AGT, AGC, TCT,TCC, TCA or TCG.

In an embodiment of the invention, a DNA sequence of human heavy-chainimmunoglobulin constant region, γ1 isotype, starting from the aminoterminus of the hinge region and ending at the carboxy-terminus of theCH3 region (stop codon underscored) comprises the nucleotide sequence:

(SEQ ID NO: 17)gttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

In an embodiment of the invention, a DNA sequence of human heavy-chainimmunoglobulin constant region, γ1 isotype, monomeric variant, startingfrom the amino terminus of the hinge region and ending at thecarboxy-terminus of the CH3 region (Cys to Ser changes underscored; stopcodon in bold font) comprises the nucleotide sequence:

(SEQ ID NO: 18)gttgagcccaaatcttctgacaaaactcacacatctccaccgtctccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

In an embodiment of the invention, a cDNA of human MCP1 (including theleader peptide) fused to mouse IgG1 comprises the nucleotide sequence:

(SEQ ID NO: 19)atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacaaaaccccgggaggagcagttcaacagcactttccgttcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttattccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatga

In an embodiment of the invention cDNA of human MCP1 (including theleader peptide) fused to human IgG4 comprises the nucleotide sequence:

(SEQ ID NO: 20)atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga

In an embodiment of the invention, a cDNA of human MCP1 (including theleader sequence) fused to human IgG4 monomeric variant comprises thenucleotide sequence:

(SEQ ID NO: 21)atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgagtccaaatatggtcccccatctccatcatctccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga

In an embodiment of the invention, a cDNA of human MCP1 (including theleader sequence) fused to human IgG1 comprises the nucleotide sequence:

(SEQ ID NO: 22)atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

In an embodiment of the invention, a cDNA of human MCP1 (including theleader sequence) fused to human IgG1 monomeric variant comprises thenucleotide sequence:

(SEQ ID NO: 23)atgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgttgagcccaaatcttctgacaaaactcacacatctccaccgtctccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

In an embodiment of the invention, a sequence of an expression plasmidencoding a fusion proteins comprising mature human MCP1 and mouse IgG,plasmid pcDNA3.1(+) hMCP1 mIgG (the initiation and the stop codonsunderscored, the codons for the linker of MCP1 and mIg in bold font),comprises the nucleotide sequence:

(SEQ ID NO: 24)gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttacgatcagtcgaattcgccgccaccatgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacaaaaccccgggaggagcagttcaacagcactttccgttcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatgactagtcatagtttagcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttattcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

The fusions of the invention comprise one or more MCP1s and one or morehalf-life extending moieties (e.g., immunoglobulins) in any order,repeated any number of times. If the fusion comprises multiple MCP1s,the MCP1s may be the same or different. For example, a fusion of theinvention comprises, in an embodiment, human MCP1-mouse MCP1-Ig.Multiple immunoglobulin polypeptides may also be included in a fusion ofthe invention. For example, in an embodiment, the fusion comprises humanMCP1-human MCP1-IgG1-IgG1; human MCP1-human MCP1-IgG1-IgG4; or humanMCP1-Ig-mouse MCP1-Ig-Ig-human MCP1. Any of these embodiments areincluded under the term “MCP1-Ig”. The present invention also includese.g., (human MCP1)₂-PEG or mouse MCP1-human MCP1-PEG

Fusions comprising MCP1 at the amino-terminus are within the scope ofthe present invention along with fusions with MCP1 at thecarboxy-terminus; the term MCP1-Ig refers to both of these types offusions. For example, the present invention comprises any of thefollowing MCP1-Ig fusions: human MCP1-Ig, Ig-human MCP1, mouse MCP1-Ig,Ig-mouse MCP1; PEG-human MCP1 or human MCP1-PEG.

In an embodiment of the invention, an MCP1-Ig fusion of the inventioncomprises a linker (e.g., a peptide linker) linking the MCP1 with theIg. In an embodiment of the invention, the linker comprises 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acids.

In addition to pcDNA3.1(+) hMCP1 mIgG, the following plasmids form partof the present invention. Plasmid pcDNA3.1(+) hMCP1 hIgG4 (theinitiation and the stop codons in underscored text, the codons for thelinker of MCP-1 and mIg in bold text):

(SEQ ID NO: 25)gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttacgatcagtcgaattcgccgccaccatgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatgactagtcatagtttagcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttaggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

Plasmid pcDNA3.1(+) hMCP1 hIgG4 monomeric variant (the initiation andthe stop codons in underscored text, the codons for the linker of MCP-1and mIg in bold text):

(SEQ ID NO: 26)gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttacgatcagtcgaattcgccgccaccatgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgagtccaaatatggtcccccatctccatcatctccagcacctgagttcctggggggaccatcagtattcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatgactagtcatagtttagcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctccagggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactattcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

Plasmid pcDNA3.1(+) hMCP1 hIgG1 (the initiation and the stop codons inunderscored text, the codons for the linker of MCP-1 and mIg in boldtext):

(SEQ ID NO: 27)gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttacgatcagtcgaattcgccgccaccatgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgactagtcatagtttagcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgattgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

Plasmid pcDNA3.1(+) hMCP1 hIgG1 monomeric variant (the initiation andthe stop codons in underscored text, the codons for the linker of MCP-1and mIg in bold text):

(SEQ ID NO: 28)gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagattacgatcagtcgaattcgccgccaccatgaaagtctctgccgcccttctgtgcctgctgctcatagcagccaccttcattccccaagggctcgctcagccagatgcaatcaatgccccagtcacctgctgttataacttcaccaataggaagatctcagtgcagaggctcgcgagctatagaagaatcaccagcagcaagtgtcccaaagaagctgtgatcttcaagaccattgtggccaaggagatctgtgctgaccccaagcagaagtgggttcaggattccatggaccacctggacaagcaaacccaaactccgaagactggatccgttgagcccaaatcttctgacaaaactcacacatctccaccgtctccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgactagtcatagtttagcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

Therapeutic Compositions and Methods

The present invention includes methods for treating or preventing anymedical condition or disorder or disease that is treatable by decreasingthe migration of chemokine receptor-bearing cells (e.g., CCR2-bearingcells; such as monocytes, macrophages, and memory T lymphocytes) intoinflammatory tissues, by decreasing endogenous chemokine (e.g., MCP1)expression or associated activity (e.g., CCR2 receptor binding), or bydecreasing the expression or activity of the chemokine receptor (e.g.,CCR2). In an embodiment of the invention, inflammatory disorders aretreated by desensitizing chemokine receptor (e.g., CCR2) bearing cells,in a subject, to the presence of the cognate chemokine ligand (e.g.,MCP1) by systemic administration of the ligand to the subject. Thechemokine ligand can be administered, in an embodiment of the invention,for a prolonged period of time so as to reach the fullest levels ofdesensitization in the cells. For example, the present inventionincludes a method for treating or preventing an inflammatory disorder ina subject by administering, to the subject, a therapeutically effectiveamount of a chemokine or multimer or fusion thereof, e.g., MCP1, an MCP1multimer or a fusion thereof (e.g., fused to an in vivo half-lifeextending moiety (e.g., PEG or Ig)) or a pharmaceutical compositionthereof (e.g., comprising a pharmaceutically acceptable carrier)optionally in association with a therapeutically effective amount of afurther therapeutic agent.

A pharmaceutical composition of the invention may be prepared by anymethods well known in the art of pharmacy; see, e.g., Gilman, et al.,(eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition, (1990), Mack Publishing Co., Easton, Pa.; Avis, et al.,(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral MedicationsDekker, New York; Lieberman, et al., (eds.) (1990) Pharmaceutical DosageForms: Tablets Dekker, New York; and Lieberman, et al., (eds.) (1990),Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York.

The term “inflammatory disorder” or “medical inflammatory disorder”includes, in an embodiment of the invention, psoriasis (e.g., nailpsoriasis, scalp psoriasis, plaque psoriasis, pustular psoriasis,guttate psoriasis, inverse psoriasis, erythrodermic or psoriaticarthritis), ankylosing spondylitis, appendicitis, peptic ulcer, gastriculcer and duodenal ulcer, peritonitis, pancreatitis, inflammatory boweldisease, colitis, ulcerative colitis, pseudomembranous colitis, acutecolitis, ischemic colitis, diverticulitis, epiglottitis, achalasia,cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease(e.g., ileocolitis, ileitis, gastroduodenal Crohn's disease,jejunoileitis or Crohn's (granulomatous) colitis), enteritis, Whipple'sdisease, asthma, allergy, anaphylactic shock, immune complex disease,organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis,vaginitis, prostatitis, and urethritis, bronchitis, emphysema, rhinitis,cystic fibrosis, pneumonitis, adult respiratory distress syndrome,pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, dermatitis, atopic dermatitis,dermatomyositis, sunburn, urticaria warts, wheals, stenosis, restenosis,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, meningitis, encephalitis,multiple sclerosis, neuritis, neuralgia, uveitis (e.g., anterior,posterior, intermediate or diffuse), arthritides and arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis (e.g., polyarticular-course juvenile rheumatoidarthritis or psoriatic arthritis), synovitis, myasthenia gravis,thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome,Behcets's syndrome, allograft rejection or graft-versus-host disease.

The present invention also includes a method for treating or preventinga parasitic, viral or bacterial infection in a subject by administering,to the subject, a therapeutically effective amount of chemokine,multimer or fusion thereof, for example, MCP1, an MCP1 multimer or afusion thereof (e.g., fused to an in vivo half-life extending moiety(e.g., PEG or Ig)) or a pharmaceutical composition thereof (e.g.,comprising a pharmaceutically acceptable carrier) optionally inassociation with a therapeutically effective amount of a furthertherapeutic agent. In an embodiment of the invention, the infection isherpes simplex virus infection (e.g., HSV1 or HSV2), human Tlymphotropic virus (HTLV; e.g., type I) infection, HIV infection, HIVneuropathy, meningitis, hepatitis (A, B or C, or the like), septicarthritis, peritonitis, pneumonia, epiglottitis, E. coli 0157:h7,hemolytic uremic syndrome, thrombolytic thrombocytopenic purpura,malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shocksyndrome, streptococcal myositis, gas gangrene, mycobacteriumtuberculosis, mycobacterium avium intracellulare, pneumocystis cariniipneumonia, pelvic inflammatory disease, orchitis, epidydimitis,legionella, lyme disease, influenza a, epstein-barr virus,vital-associated hemaphagocytic syndrome, vital encephalitis or asepticmeningitis.

The present invention also includes a method for treating or preventinga cancer or malignancy (e.g., breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, colorectal, thyroid, pancreatic,prostate or bladder cancer) in a subject by administering, to thesubject, a therapeutically effective amount of chemokine, multimer orfusion thereof, for example, MCP1, an MCP1 multimer or a fusion thereof(e.g., fused to an in vivo half-life extending moiety (e.g., PEG or Ig))or a pharmaceutical composition thereof (e.g., comprising apharmaceutically acceptable carrier) optionally in association with atherapeutically effective amount of a further therapeutic agent.

The present invention also includes a method for treating or preventinga cardiovascular or circulatory disorder in a subject by administering,to the subject, a therapeutically effective amount of chemokine,multimer or fusion thereof, for example, MCP1, an MCP1 multimer or afusion thereof (e.g., fused to an in vivo half-life extending moiety(e.g., PEG or Ig)) or a pharmaceutical composition thereof (e.g.,comprising a pharmaceutically acceptable carrier) optionally inassociation with a therapeutically effective amount of a furthertherapeutic agent. In an embodiment of the invention, the disease ordisorder is cardiac stun syndrome, myocardial infarction, congestiveheart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis,atherosclerosis, restenosis, diabetic ateriosclerotic disease,hypertension, arterial hypertension, renovascular hypertension, syncope,shock, syphilis of the cardiovascular system, heart failure, corpulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrialectopic beats, atrial flutter, atrial fibrillation (sustained orparoxysmal), post perfusion syndrome, cardiopulmonary bypassinflammation response, chaotic or multifocal atrial tachycardia, regularnarrow QRS tachycardia, specific arrythmias, ventricular fibrillation,His bundle arrythmias, atrioventricular block, bundle branch block,myocardial ischemic disorders, coronary artery disease, angina pectoris,myocardial infarction, cardiomyopathy, dilated congestivecardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,endocarditis, pericardial disease, cardiac tumors, aordic and peripheralaneurysms, aortic dissection, inflammation of the aorta, occlusion ofthe abdominal aorta and its branches, peripheral vascular disorders,occlusive arterial disorders, peripheral atherlosclerotic disease,thromboangitis obliterans, functional peripheral arterial disorders,Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venousdiseases, venous thrombosis, varicose veins, arteriovenous fistula,lymphederma, lipedema, unstable angina, reperfusion injury, post pumpsyndrome or ischemia-reperfusion injury.

A pharmaceutical composition containing chemokine, multimer or fusionthereof, for example, MCP1 or a multimer or fusion thereof (e.g.,MCP1-Ig) can be prepared using conventional pharmaceutically acceptableexcipients and additives and conventional techniques. Suchpharmaceutically acceptable excipients and additives include non-toxiccompatible fillers, binders, disintegrants, buffers, preservatives,anti-oxidants, lubricants, flavorings, thickeners, coloring agents,emulsifiers and the like. All routes of administration are contemplatedincluding, but not limited to, parenteral (e.g., subcutaneous,intravenous, intraperitoneal, intratumoral or intramuscular) andnon-parenteral (e.g., oral, transdermal, intranasal, intraocular,sublingual, inhalation, rectal and topical).

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Theinjectables, solutions and emulsions can also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations are generally added to parenteralpreparations packaged in multiple-dose containers which include phenolsor cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN-80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

Preparations for parenteral administration can include sterile solutionsready for injection, sterile dry soluble products, such as lyophilizedpowders, ready to be combined with a solvent just prior to use,including hypodermic tablets, sterile suspensions ready for injection,sterile dry insoluble products ready to be combined with a vehicle justprior to use and sterile emulsions. The solutions may be either aqueousor nonaqueous.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained is also contemplated herein.Briefly, in this embodiment, chemokine, multimer or fusion thereof, forexample, MCP1 or a fusion or multimer thereof (e.g., MCP1-Ig) isdispersed in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient diffuses through the outer polymericmembrane in a release rate controlling step. The percentage of activecompound contained in such parenteral compositions is highly dependenton the specific nature thereof, as well as the activity of thechemokine, multimer or fusion thereof, for example, MCP1 or fusion ormultimer thereof and the needs of the subject.

The concentration of the chemokine, multimer or fusion thereof, forexample, MCP1 or fusion or multimer thereof can be adjusted so that aninjection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends, inter alia, on the age,weight and condition of the patient or animal as is known in the art.

A unit-dose parenteral preparation comprising chemokine, multimer orfusion thereof, for example, MCP1 or fusion or multimer thereof ispackaged in an ampoule, a vial or a syringe with a needle. Allpreparations for parenteral administration must be sterile, as is knownand practiced in the art. Such a preparation forms part of the presentinvention.

A chemokine, multimer or fusion thereof, for example, MCP1 or a fusionor multimer thereof can be formulated into a lyophilized powder, whichcan be reconstituted for administration as solutions, emulsions andother mixtures. The powder may also be reconstituted and formulated as asolid or gel.

The sterile, lyophilized powder is prepared by dissolving chemokine,multimer or fusion thereof, for example, MCP1 or a fusion or multimerthereof, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. The solvent may contain an excipient which improvesthe stability or another pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial can contain a single dosage or multipledosages of the chemokine, multimer or fusion thereof, for example, theMCP1 or fusion or multimer thereof. The lyophilized powder can be storedunder appropriate conditions, such as at about 4° C. to roomtemperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder can be added to sterile water oranother suitable carrier. The precise amount depends upon the selectedcompound. Such an amount can be empirically determined.

Administration by inhalation can be provided by using, e.g., an aerosolcontaining sorbitan trioleate or oleic acid, for example, together withtrichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethaneor any other biologically compatible propellant gas; it is also possibleto use a system containing chemokine, multimer or fusion thereof, forexample, MCP1 or fusion or multimer thereof, by itself or associatedwith an excipient, in powder form.

In an embodiment, chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof is formulated into a solid dosageform for oral administration, in one embodiment, into a capsule ortablet. Tablets, pills, capsules, troches and the like can contain oneor more of the following ingredients, or compounds of a similar nature:a binder; a lubricant; a diluent; a glidant; a disintegrating agent; acoloring agent; a sweetening agent; a flavoring agent; a wetting agent;an emetic coating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The scope of the present invention includes methods comprisingadministration of chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof, or a pharmaceutical compositionthereof, in association with, for example, one or more furthertherapeutic agents as well as compositions comprising chemokine,multimer or fusion thereof, for example, MCP1 or a fusion or multimerthereof in association with a further therapeutic agent. In anembodiment, the other therapeutic agent is an agent that, whenadministered to a subject, treats or prevents an inflammatory conditionin the subject. The administration and dosage of any such agent istypically as according to the schedule listed in the product informationsheet of the approved agents, in the Physicians' Desk Reference 2003(Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN:1563634457; 57th edition (November 2002), as well as therapeuticprotocols well known in the art.

The term “in association with” indicates that the chemokine, multimer orfusion thereof, for example, the MCP1 or a fusion or multimer thereofand the further therapeutic agent can be formulated into a singlecomposition for simultaneous delivery or formulated separately into twoor more compositions (e.g., a kit). Furthermore, each component can beadministered to a subject at a different time than when the othercomponent is administered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at severalintervals over a given period of time. Moreover, the separate componentsmay be administered to a subject by the same or by a different route(e.g., orally, intravenously, subcutaneously).

A “further therapeutic agent” is any agent, other than the chemokine,multimer or fusion thereof, that, when administered to a subject, bringsabout a desired or beneficial therapeutic effect, such as prevention,elimination or reduction of the progression or severity of symptomsassociated with a given medical condition (e.g., an inflammatorydisorder). A further therapeutic agent may be, for example, ananti-inflammatory agent or a pain reliever.

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more non-steroidalanti-inflammatory drug (NSAIDs) such as aspirin, diclofenac, diflunisal,etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid,meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam,salsalate, sulindac, tenoxicam, tiaprofenic acid or tolmetin.

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more topicalmedications, for example, anthralin, calcipotriene, salicylic acid, coaltar, tazarotene, topical steroids (e.g., Clobetasol propionate;Clobetasol propionate; Betamethasone dipropionate; Clobetasolpropionate; Diflorasone diacetate; Clobetasol propionate Halobetasolpropionate; Amcinonide; Betamethasone dipropionate; Betamethasonedipropionate; Mometasone furoate; Diflorasone diacetate; Halcinonide;Fluocinonide; Diflorasone diacetate; Betamethasone dipropionate;Diflorasone diacetate; Desoximetasone; Desoximetasone; Triamcinoloneacetonide; Fluticasone propionate; Amcinonide; Betamethasonedipropionate; Diflorasone diacetate; Fluocinonide; Betamethasonevalerate; Diflorasone diacetate; Betamethasone dipropionate;Desoximetasone; Betamethasone valerate; Triamcinolone acetonide;Flurandrenolide; Fluocinolone acetonide; Mometasone furoate;Triamcinolone acetonide; Fluocinolone acetonide; Betamethasone benzoate;Hydrocortisone valerate; Flurandrenolide; Fluticasone propionate;Prednicarbate; Desonide; Betamethasone dipropionate; Triamcinoloneacetonide; Hydrocortisone; Fluocinolone acetonide; Betamethasonebenzoate; Betamethasone valerate; Hydrocortisone valerate; Alclometasonedipropionate; Desonide; Fluocinolone acetonide; Desonide; Betamethasonevalerate; or a mixture of hydrocortisone, dexamethasone,methylprednisolone and prednisolone), petroleum jelly, aloe vera,oilated oatmeal, epsom salts or Dead Sea salts.

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more of alefacept,etanercept, cyclosporine, methotrexate, acitretin, isotretinoin,hydroxyurea, mycophenolate mofetil, sulfasalazine or 6-Thioguanine.

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more of anakinra,injectable gold, penicillamine, azathioprine, chloroquine,hydroxychloroquine, sulfasalazine or oral gold (e.g., auranofin, goldsodium thiomalate or aurothioglucose).

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more of mesalamine,sulfasalazine, budesonide, metronidazole, ciprofloxacin, azathioprine,6-mercaptopurine or dietary supplementation of calcium, folate orvitamin B12.

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more COX2 inhibitorssuch as celecoxib (Celebrex®), rofecoxib (Vioxx®), valdecoxib (Bextra®),lumiracoxib (Prexige™) or etoricoxib (Arcoxia®).

Further therapeutic agents that may be administered or combined inassociation with the chemokine, multimer or fusion thereof, for example,MCP1 or a fusion or multimer thereof include one or more antibodies suchas efalizumab (Raptiva®), adalimumab (Humira®), infliximab (Remicade®)or ABX-IL8.

In an embodiment of the invention, the chemokine, multimer or fusionthereof, for example, MCP1 or a fusion or multimer thereof isadministered, to a subject, in association with phototherapy,particularly, wherein the subject suffers from psoriasis. In such anembodiment, the subject is exposed to sunlight, UVB light, PUVA(psoralen plus ultraviolet A). PUVA (psoralin-UVA) or laser light. PUVAuses ultraviolet A light to treat psoriasis in combination withpsoralen, an oral or topical medication that makes your skin moresensitive to light. Lasers emit highly focused beams of light thataffects primarily the psoriatic skin while healthy skin isn't exposedsignificantly. One type of laser, the XTRAC excimer laser, uses highlyfocused ultraviolet B light. Another type of laser used for psoriasis isa pulsed dye laser, which uses pulses of yellow light—different from theultraviolet rays used in UVB or XTRAC—to destroy some of the blood cellsthat grow in patches of psoriasis. Treatment with pulsed dye lasersusually takes a few months, with appointments every three weeks.

Dosage and Administration

Typical protocols for the therapeutic administration of a composition ofthe invention are well known in the art. Pharmaceutical compositions ofthe invention may be administered, for example, by any parenteral (e.g.,subcutaneous injection, intramuscular injection, intravenous injection)or non-parenteral route (e.g., orally, nasally).

Pills and capsules of the invention can be administered orally.Injectable compositions can be administered with medical devices knownin the art; for example, by injection with a hypodermic needle.

Injectable pharmaceutical compositions of the invention may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824 or 4,596,556.

In an embodiment, the daily dose of a “further therapeutic agent” (e.g.,an anti-inflammatory agent) administered in association with thechemokine, multimer or fusion thereof, for example, MCP1 or a fusion ormultimer thereof is, where possible, administered accordance with thePhysicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed);Medical Economics Company; ISBN: 1563634457; 57th edition (November2002). The proper dosage can, however, be altered by a clinician tocompensate for particular characteristics of the subject receiving thetherapy depending, for example, on the potency of the compoundadministered or of the chemokine, multimer or fusion thereof (e.g.,MCP1-Ig), side-effects, age, weight, medical condition, overall healthand response.

The present invention provides methods for treating or preventing aninflammatory condition in a subject by administering, to the subject, atherapeutically effective amount of chemokine, multimer or fusionthereof, for example, MCP1 or a fusion or multimer thereof, optionallyin association with a therapeutically effective amount of a furthertherapeutic agent. The term “therapeutically effective amount” meansthat amount of a therapeutic agent or substance (e.g., MCP1-Ig) thatwill elicit a biological or medical response of a tissue, system,subject or host that is being sought by the administrator (such as aresearcher, doctor or veterinarian) which includes, for example,alleviation, reversal, elimination or halting or slowing of progressionof a target medical disorder or any symptom thereof to any degreeincluding prevention of the disorder in the subject. In an embodiment ofthe invention, a therapeutically effective amount or dosage ofchemokine, multimer or fusion thereof, for example, MCP1 or a fusion ormultimer thereof (e.g., MCP1-Ig; for example a polypeptide comprisingany amino acid sequence as set forth in SEQ ID NO: 8, 9, 10, 11 or 12)is from about 0.1 mpk (mg per kilogram of body weight) to about 10 mpk(e.g., 0.25, 0.5, 0.75 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mpk) once a day,every 2 days, every 4 days or every 5 days or once a week.

A composition of the invention can be administered, for example, threetimes a day, twice a day, once a day, three times weekly, twice weeklyor once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8 weeks.Moreover, the composition can be administered over a short or longperiod of time (e.g., 1 week, 1 month, 1 year, 5 years)

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, the dose can be reduced orincreased as indicated by exigencies of the therapeutic situation. Forexample, dosage can be adjusted, by a practitioner of ordinary skill inthe art (e.g., physician or veterinarian) according to the drug'sefficacy, progression or persistence of the disease or any of itssymptoms or the patient's age, weight, height, past medical history,present medications and the potential for cross-reaction, allergies,sensitivities and adverse side-effects.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of chemokine, multimer or fusion thereof, for example, MCP1or a fusion or multimer thereof or a pharmaceutical composition thereofat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

For example, psoriasis progress can be monitored, by the physician orveterinarian by a variety of methods, and the dosing regimen can bealtered accordingly. Methods by which to monitor psoriasis include, forexample, by skin biopsy, or scraping and culture of skin patches,monitoring the spread of the condition on the skin of the subject or byan X-ray to check for psoriatic arthritis if joint pain is present andpersistent.

For example, rheumatoid arthritis progress can be monitored, by thephysician or veterinarian, by a variety of methods, and the dosingregimen can be altered accordingly. Methods by which to monitorrheumatoid arthritis include, for example, joint X-rays, a rheumatoidfactor blood test, checking for elevated erythrocyte sedimentation rate(ESR), a complete blood count to check for low hematocrit (anemia) orabnormal platelet counts, a blood test to check for C-reactive proteinor synovial fluid analysis.

For example, Crohn's disease progress can be monitored, by the physicianor veterinarian, by a variety of methods, and the dosing regimen can bealtered accordingly. Methods by which to monitor Crohn's diseaseinclude, for example, monitoring the severity of symptoms reported bythe subject or patient, sigmoidoscopy, colonoscopy, ERCP (endoscopicretrograde cholangiopancreatography), endoscopic ultrasound, capsuleendoscopy, Plain X-rays, X-rays with Contrast, CT Scan or white bloodcell scan.

For example, uveitis progress can be monitored, by the physician orveterinarian, by a variety of methods, and the dosing regimen can bealtered accordingly. Methods by which to monitor uveitis include, forexample, examination of the eye with a slit lamp microscope andophthalmoscopy, measuring visual acuity and intraocular pressure.

For example, ulcerative colitis progress can be monitored, by thephysician or veterinarian, by a variety of methods, and the dosingregimen can be altered accordingly. Methods by which to monitorulcerative colitis include, for example, routine check-ups,colonoscopies, rectal or colon biopsy, stool testing for blood or pus,blood tests to examine white blood cell levels or X-ray examination.

EXAMPLES

The following examples are provided to more clearly describe the presentinvention and should not be construed to limit the scope of theinvention. Any method or composition disclosed in the Examples sectionconstitute part of the present invention.

Example 1 Design, Construction, Expression and Purification of HumanMCP1-Mouse Ig Heavy Chain γ1 (hinge-CH2-CH3) Fusion

Design of Constructs.

hMCP1-mIg (NH₂-human MCP1-Mouse Ig Heavy chain γ1 (hinge-CH2-CH3)-COOH):A BamH1 site was introduced as a linker in the cDNA, resulting in aninsertion of a dipeptide, Gly-Ser, at the junction of the MCP1 and theIg H chain. The product was expected to form a dimer with predictedmolecular mass of 68,993.

hMCP1-hIgG4 (NH₂-human MCP1-human Ig H chain γ4 (hinge-CH2-CH3)-COOH): ABamH1 site was introduced as a linker in the cDNA, resulting in aninsertion of a dipeptide, Gly-Ser, at the junction of the MCP1 and theIg H chain. The product was expected to form a dimer with predictedmolecular mass of 69,146.

hMCP1-hIgG4 monomeric variant (NH₂-human MCP1-human Ig Heavy chain γ4(hinge-CH2-CH3)-COOH): The two cysteine residues were replaced by serineresidues to eliminate the intermolecular disulfide bonds. A BamH1 sitewas introduced as a linker in the cDNA, resulting in an insertion of adipeptide, Gly-Ser, at the junction of the MCP1 and the Ig H chain. Theproduct was expected to form a dimer with predicted molecular mass of34,543.

hMCP1-hIgG1 (NH₂-human MCP1-human Ig H chain γ1 (hinge-CH2-CH3)-COOH): ABamH1 site was introduced as a linker in the cDNA, resulting in aninsertion of a dipeptide, Gly-Ser, at the junction of the MCP1 and theIg H chain. The product was expected to form a dimer with predictedmolecular mass of 70,000.

hMCP1-hIgG1 monomeric variant (NH₂-human MCP1-human Ig Heavy chain γ1(hinge-CH2-CH3)-COOH): The three cysteine residues were replaced byserine residues to eliminate the intermolecular disulfide bonds. A BamH1site was introduced as a linker in the cDNA, resulting in an insertionof a dipeptide, Gly-Ser, at the junction of the MCP1 and the Ig H chain.The product was expected to form a dimer with predicted molecular massof 34,955.

Expression and purification. In this example, MCP1-Ig was expressed inmammalian cells, secreted and then isolated from the cellular growthmedia. The isolated protein was analyzed by SDS-PAGE analysis.

The cDNA of human MCP1 (see Genbank accession No. NM_(—)002982) and apartial cDNA derived from the constant region of mouse Ig (see Genbankaccession No. BC057688) (including the coding sequences for the hinge,the CH2, and the CH3 regions) were cloned by reverse-transcriptionpolymerase chain reaction (RT-PCR). The MCP1-Ig cDNA was cloned intomammalian expression vector pcDNA3.1 (+) (Invitrogen, Carlsbad, Calif.)as a Hind3-Not1 fragment by standard molecular biology procedures tocreate the pcDNA3.1(+)hMCP1 mIgG plasmid.

CHO-K1 (ATCC CRL-9618) cells were maintained in D-MEM/F-12 medium(Invitrogen, Carlsbad, Calif.) supplemented with 5% fetal bovine serum).The plasmid DNA was introduced into CHO-K1 cells using the Lipofectamine2000 transfection kit (Invitrogen, Carlsbad, Calif.) by following theprotocol suggested by the manufacturer. At forty-eight hourspost-transfection, G418 (Invitrogen) was added to the culture at 1 mg/mlfor selection of stably transfected cells. The G418 resistant cellsappeared as colonies in about 10-14 days post transfection. The cellswere then pooled and transfected by limiting dilution into 96-welltissue culture plates at frequencies of 3 cells per well, 1 cells perwell, and 0.3 cells per well. Single cell-derived clones appeared inabout 7-10 days and the conditioned media were examined by enzyme-linkedimmunosorbent assay (ELISA) specific for mouse IgG1 (Bethyl, Montgomery,Tex.). Clones giving highest titers were re-examined for expressionlevel by normalizing the yield to the cell numbers. A clone producing atgreater than 40 mg per liter was chosen for production (clone 52).

Production of the hMCP1-mIg protein was carried out under serum-freeconditions. Clone 52 cells were stepwisely weaned into suspensionculture of protein-free medium consisting of IS-CHO V base medium(Irvine Scientific, Irvine, Calif.). Each liter of the protein-freemedium contains 8 mM glutamine (Invitrogen, Gaithersburg, Md.), 10 ml of100×HT (Invitrogen), 1 ml of CD-lipid (Invitrogen), 8 ml of 45% glucose(Sigma, St. Louis, Mo.), 20 ml of GSEM (Sigma), 1 ml each of TraceElement A and Trace Element B (Cellgro, Herndon, Va.). For production,cells were seeded at a density of 0.5×10⁶ per ml in a volume of 1 literin a 3-liter shaker flask. The flask was shaken at a speed of 75 rpm ata constant temperature of 37° C. in the presence of 7.5% CO₂. Theconcentration of glutamine was maintained at about 300 milligrams perliter and the concentration of glucose was maintained at 1-2 gram perliter. Conditioned media were harvested at approximately 14 days whencell viability was about 20%. The conditioned media were filteredthrough a 2-micron filtration unit followed by processing through anAffi-gel protein-A affinity column (BioRad, Hercules, Calif.) accordingto the manufacturer's suggested protocol. The purified protein wasanalyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) underreducing conditions. A single band was observed after the gel wasstained by SimplyBlue SafeStain (Invitrogen), with an estimated size ofabout 34 kilodaltons. The purity of the product was estimated greaterthan 99%.

Example 2 Cell Migration Assay

In this example, the presence of human MCP1-mIg was demonstrated toimpede the ability of THP-1 human monocytic cells to migrate toward arecombinant human MCP1 gradient was demonstrated.

THP-1 cells (ATCC TIB202) were maintained in RPMI1640 supplemented with10% fetal bovine serum, 1 mM sodium pyruvate, 4.5 g/liter glucose, 1.5g/l sodium bicarbonate, 10 mM HEPES, 0.05 mM beta-mercaptoethanol, andpenicillin/streptomycin. Cell migration assay was performed using96-well ChemoTx microplates with a 5 μm filter (NeuroProbe, Gathersburg,Md.) according to the manufacturer's instructions. Recombinant humanMCP1 (rhMCP1) (R & D Systems, Minneapolis, Minn.) was placed in thebottom chamber. hMCP1-mIg or isotype control IgG was placed in both thetop and the bottom chambers. Cells were dispensed in the top wells. Themicroplates were placed in a 37° C. humidified CO₂ (5%) incubator for 2hours to allow the cells to migrate toward human MCP1 in the bottomchamber. Cell migration was quantitated as relative luminescent units(RLU) by CellTiter-Glo Luminiscent Cell Viability Assay Kit (Promega,Madison, Wis.) according to the manufacturer's protocol. Cell migrationwas calculated by subtracting the RLU of spontaneous migration from theRLU of the migration in the presence of the chemokine reagents. Therelative % of migration was calculated by using the highest number ofcell migration as 100%.

The approximated EC50 values for hMCP1-mIg and rhMCP1 were about 0.5 nMand 0.05 nM, respectively (Table 1, left and middle columns). hMCP1-mIgat 3 nM caused a significant reduction of the ability of THP-1 cells tomigrate toward a gradient of rhMCP1 (Table 1, right column).

TABLE 1 Effects of hMCP1-mlg and recombinant human MCP1 (rhMCP1) on themigration of THP-1 human monocytic cells. Relative % of cell migrationhMCP1-mlg rhMCP1 rhMCP1 plus Conc. (nM) alone alone hMCP1-mlg at 3 nM0.01 8.3 41 −3 0.03 14.2 49.2 5.8 0.1 29.8 59.4 8.7 0.3 30.0 72.0 −0.2 159.0 90.5 7.6 3 81.2 100 34.1 10 73.0 58.6 35.5 30 96.6 24.8 3.5 10075.5 −18.3 −10.7

Example 3 Collagen-Induced Arthritis Assay

Male B10.RIII mice, 12-13 weeks of age, were immunized with bovine typeII collagen (BCII) (Elastin Products, Owensville, Mo.), Difco incompleteFreunds adjuvant and Mycobacteria tuberculosis (MBT, strain H37RA,Sigma, St. Louis, Mo.). BCII was dissolved overnight at 4° C. in 0.01 Macetic acid (60 mg BCII in 25 ml 0.01 M acetic acid). Complete Freundsadjuvant (CFA) was prepared by mixing M. tuberculosis with Difcoincomplete Freunds adjuvant (1 mg/ml). One volume of BCII and one volumeof CFA were then mixed to emulsify. The emulsion contained 1200 μg/mlBCII and 0.5 mg/ml M. tuberculosis.

Mice were immunized by intradermal injection at 5 sites on the backincluding 1 site at the base of the tail. Each mouse received a totalvolume of 0.25 ml of the emulsion, which is equivalent to 300 μgBCII/mouse.

Mice were scored for symptoms and severity of arthritis 16 days afterthe immunization and divided into groups of 15 for dosing such that eachgroup has the same number of mice with arthritis. Scores were based on ascale of 0 to 4 as follows:

0=normal

1=redness

2=one or more digits swollen

3=entire paw swollen

4=ankylosis

Scores were taken 4 days before the boost and on days 0, 2, 4, 7 & 9.Swelling was also evaluated on these days by caliper measurement.

The immunizations were also boosted: BCII was dissolved overnight in0.01M acetic acid (12.5 mg BCII in 25 ml 0.01M acetic acid). Mice wereboosted, ip, with 100 μg BCII/0.2 ml/mouse, 20 days after immunization(Day 0 based on the boost).

hMCP1-mIg (SEQ ID NO: 8) and mIgG1 (TC31-27F11, SP-BioPharma, Palo Alto,Calif.) were prepared in phosphor-buffered saline at 2 mg/ml andadministered intraperitoneally to the mice at indicated time points. Thefirst dose was given at 20 milligrams per kilogram body weight (mpk).The subsequent doses were given at 10 mpk.

Serum samples were taken at the termination for determination ofanti-collagen antibody (IgG2a).

A protecting effect was observed in the group of hMCP1-mIg-treated miceas indicated by the mean disease scores of Table 2 as well as by the pawswelling values of Table 3.

Plasma levels of anti-collagen IgG2a were determined by ELISA. Theplates were coated with ELISA grade Type II Bovine Collagen (Chondrex,Redmond, Wash.) at 50 μl per ml at 4° C. overnight. The plate was washedby PBS followed by blocking with 1% BSA at 4° C. overnight. After abrief wash, samples were diluted at 1:10,000 and applied to the platealong with the standards (US Biologicals, Swampscott, Mass.) of 2-foldserial dilutions starting at 40 pg per ml. The plate was incubated at 4°C. overnight. After wash 100 μl of Peroxidase-conjugated anti-IgG(Abcam, Cambridge, Mass.) (1:500 diluted) was added to the wells andincubated at room temperature for 2 hours. The plate was washed and 100μl of TMB (Sigma) was added to the wells. The plate was read at 490 nM.

In hMCP1-mIg treated animals, the levels of anti-collagen IgG2a antibodywere suppressed, as compared to the levels of the isotype control(mIgG1)-treated animals (Table 4).

TABLE 2 Arthritic score of collagen arthritis in mice (mean ± SEM).Scores Day Day Day Day Day Day −4 0 2 4 7 9 Normal 0 0 0 0 0 0 Isotypectrl 0 0.6 ± 0.3 2.4 ± 0.7  3.1 ± 0.7  5.2 ± 0.8  5.2 ± 0.8 (mlgG1)hMCP1- 0 0.1 ± 0.1 0.8 ± 0.3* 1.1 ± 0.4* 2.7 ± 0.5* 3.5 ± 0.7 mlg *p <0.05.

TABLE 3 Paw swelling of collagen arthritis in mice (mean ± SEM). Pawswelling (mm) Treatment Day −4 Day 0 Day 2 Day 4 Day 7 Day 9 Normal 1.80± 0.00 1.80 ± 0.00 1.80 ± 0.00 1.82 ± 0.01 1.83 ± 0.01 1.83 ± 0.02Isotype ctrl 1.80 ± 0.00 1.83 ± 0.03 1.88 ± 0.04 1.99 ± 0.05 2.15 ± 0.072.23 ± 0.08 (mlgG1) hMCP1-mlg 1.80 ± 0.00 1.81 ± 0.00  1.80 ± 0.00* 1.82 ± 0.01*  1.89 ± 0.04*  2.03 ± 0.06* *p < 0.05 of the paw sizes ofthe hMCP1-mlg-treated comparing to those of the isotype control group.

TABLE 4 Plasma levels of anti-collagen IgG_(2a) at day 9. Plasma levels(μg/ml) Treatment of anti-collagen IgG_(2a) Normal 731.40 ± 30.91Isotype control (mlgG1) 100554 ± 25576 hMCP1-mlg  51738 ± 13880Data were expressed as mean with standard error.

Example 4 Receptor Binding Assay

In this example, the ability of hMCP1 (R & D Systems) and hMCP1-mIg (SEQID NO: 8) to bind the CCR2 receptor was assayed.

‘Full-length human CCR2 cDNA (Genbank Accession No. NM_(—)000648) wasgenerated from human peripheral blood mononuclear cells by RT-PCR usingoligonucleotide sequence derived from a published sequence. Full-lengthmouse CCR2 cDNA (Genbank Accession No. NM_(—)009915) was also generatedfrom mouse splenocytes by RT-PCR using oligonucleotide sequence derivedfrom a published sequence.

Murine IL-3 dependent pro-B cells Ba/F3 were maintained in RPMI 1640medium (Invitrogen, Gaithersburg, Md.) supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 100 μg per ml streptomycin, and 100 ugper ml penicillin, 50 μM 2-mercaptoethanol and 2 μg per ml ofrecombinant mouse IL-3 (Biosource International, Camarillo, Calif.).Recombinant Ba/F3 cells expressing human CCR2 and mouse CCR2 wereestablished by stably transfecting Ba/F3 mouse pre-B cells withpME18Sneo-hCCR2 or mCCR2 plasmids by electroporation with a protocoldescribed in Chou et al. British J. Pharmacology 137:663 (2002). Stabletransfectants were selected in the presence of Geneticin (Invitrogen) at1 mg/ml.

Cell membranes were prepared as previously described in Chou et al.,2002. Briefly, cells were pelleted, resuspended in a lysis buffer (10 mMHEPES, pH 7.5 and Complete® protease inhibitors (Boehringer Mannheim,Indianapolis, Ind.) and incubated on ice for 5 min. The cells weretransferred to a 4639 cell disruption bomb (Parr Instrument, Moline,Ill.) and disrupted with 1500 psi nitrogen for 30 min on ice. Followingremoval of large cellular debris by centrifugation at 500 g for 5 min,cell membranes in the supernatant were pelleted by centrifugation at100,000 g for 30 min. Membranes were resuspended in lysis buffercontaining 10% sucrose and stored at −80° C. Radiolabeled human MCP1(specific activity=2200 Ci/mmol) was purchased from Perkin-Elmer(Boston, Mass.).

In CCR2 receptor binding assay, the binding reaction was carried outunder the following conditions: 50 mM HEPES, 10 mM NaCl, 1 mM CaCl₂, 10mM MgCl₂, 0.1% bovine serum albumin, 2 μg cell membrane and 160 μgwheat-germ agglutinin SPA beads (Amersham, Piscataway, N.J.), 30 pMradioiodinated human MCP1, and competing agents at indicatedconcentrations. The reaction mixtures were incubated at room temperaturewith constant rocking. Membrane-bound radiolabeled rhMCP1 was measuredusing a 1450 Microbeta Trilux counter (Wallac, Gaithersburg, Md.). TheEC₅₀ and the Ki values were calculated using the GraphPad Prism 4software (San Diego, Calif.).

hMCP1-mIg competed with radiolabeled rhMCP1 in binding to the membranesof cells containing CCR2, with a potency of 5-12 fold less thanunlabeled rhMCP1 (Table 5).

TABLE 5 Inhibition of CCR2 binding of radio-iodinated rhMCP1 Ba/F3-hCCR2cells Ba/F3-mCCR2 cells IC₅₀ (pM) K_(i) (pM) IC₅₀ (pM) K_(i) (pM) rhMCP1183.0 84.5 29.8 11.8 hMCP1-mlg 623.9 287.5 350.2 138.2

Example 5 Expression, Purification and Characterization of MCP1-IgFusions

In this example, fusions were expressed and purified and characterized.

Variants of hMCP-1-hIg fusion proteins. Human MCP1-hIg variant proteinswere expressed in mammalian cells, secreted and then isolated from thecellular growth media. Purification was conducted using the sameprotocols as set forth above for purifying hMCP-1-mIg. The isolatedproteins were analyzed by SDS-PAGE.

The cDNA of human MCP1 (see Genbank accession No. NM 002982) and apartial cDNA derived from the constant region of human immunoglobulinheavy chain gamma 1 isotype (see Genbank accession No. 019046) or gamma4 isotype (see Genbank accession No. BC025985) (in both cases includingthe coding sequences for the hinge, the CH2, and the CH3 regions) werecloned by reverse-transcription polymerase chain reaction (RT-PCR). Tocreate monomeric forms of the gamma 1 and gamma 4 variants, the cysteineresidues in the hinge region were replaced by serine residues.

The cDNAs corresponding to the hMCP1-hIg variants were individuallycloned into mammalian expression vector pcDNA3.1 (+) (Invitrogen,Carlsbad, Calif.) as Hind3-Not1 fragment by standard molecular biologyprocedures to create the pcDNA3.1(+)hMCP1-hIgG plasmid.

The hMCP-1-hIg variants were expressed by stably transfecting thecorresponding plasmids into CHO-K1 cells. The protocols of transfection,selection of stable clones, tissue culture, and product purificationwere similar to those described in the example of hMCP-1-mIg.

The binding affinities of the hMCP-1-hIg variants to CCR2 receptor weredetermined using the membranes of THP-1 cells, as in the example ofhMCP-1-mIg. The K_(i) values of the variants were estimated to be 16.8pM for the dimeric form of hMCP-1-hIg(γ1), 28.8 pM for the monomericform of hMCP-1-hIg(γ1), 51.9 pM for the dimeric form of hMCP-1-hIg(γ4),and 90.1 pM for the monomeric form of hMCP-1-hIg(γ4). For comparison,the K_(i) value of hMCP-1 in the same experiment was determined to be65.7 pM.

The relative potencies of the variants were also determined bychemotaxis assays with THP-1 cells. The protocol was similar to thatdescribed in the example of hMCP-1-mIg. The estimated EC₅₀ values were50 pM for the dimeric form of hMCP-1-hIg(γ1), 90 pM for the monomericform of hMCP-1-hIg(γ1), 500 pM for the dimeric form of hMCP-1-hIg(γ4),and 500 pM for the monomeric form of hMCP-1-hIg(γ4). For comparison, theEC₅₀ value of hMCP-1 in the same experiment was determined to be 200-400pM.

The ability of the hMCP-1-hIg variants to desensitize CCR2 wasdetermined with THP-1 cells by chemotaxis assays. The protocol issimilar to that described in the example of hMCP-1-mIg. The cells wereincubated with each variant hMCP-1-hIg for 30 minutes prior to test formigration towards hMCP-1. With the pretreatment of either the dimeric orthe monomeric forms of hMCP-1-hIg(γ1), at as low as 1 nM of theIg-fusion proteins, the migration of THP-1 cells was completelyabolished. When the cells were preincubated with the dimeric or themonomeric forms of hMCP-1-hIg(γ4) each at 1 nM, the EC₅₀ value of hMCP-1increased by 2-4 fold. When the preincubation was carried out with thedimeric or the monomeric forms of hMCP-1-hIg(γ4) at 10 nM, the EC₅₀value of hMCP-1 increased by 30 fold or more.

Purification of hMCP-1-mIg. For large-scale purification (200 mg and up)of hMCP-1-mIg (used in EAE and anti-collagen antibody-induced arthritismodels set forth below), ProA affinity chromatography was employed usingthe rProA Sepharose FF or the MabSelect resin, from GE Healthcare(Uppsala, Sweden). The affinity column was equilibriated with a highsalt buffer consisting of sodium phosphate at 10 mM, pH 7.2, and sodiumchloride at 125 mM NaCl. Conditioned medium was loaded to the columnfollowed by a 10 bed-volumes wash with the same buffer as indicatedbefore. The sample-loaded column was then washed with 5 bed-volumes ofphosphate buffer including sodium phosphate at 10 mM, pH 7.2. Productelution was carried out with 5 bed-volumes of acetic acid at 0.1 M at pH2.9. The eluate was immediately neutralized by bringing the pH to 7.2with Tris Base at 1 M. After the pH adjustment, the pool was filteredwith Stericup Express GP Plus 0.22 μm (Millipore, Bedford Mass.).

An optional step of anion-exchange chromatography with Q SepharoseHiTRAP FF (GE Healthcare) was used as needed to remove minor impurities.The equilibration and wash buffer consisted of sodium phosphate at 10mM, pH 7.2, and sodium chloride at 125 mM NaCl. The product was in theflow-through.

For product concentrating, an Amicon Stir Cell, Model 8050 with 10Kregenerated cellulose (Millipore) was used.

Pharmacokinetics of hMCP-1-mIg. C57B6 mice were used for pharmacokineticstudies of hMCP-1-mIg. Mice in groups of three were injectedintravenously, intraperitoneally, or subcutaneously with a single doseof hMCP-1-mIg at 10 milligrams per kilogram body weight. Serum sampleswere collected at time points from 30 minutes to 7 days. The residuallevels of hMCP-1-mIg were determined by enzyme-linked immunosorbentassay (ELISA) using anti-hMCP-1 antibody (R&D Systems, Minneapolis,Minn.) as the capture antibody and horse-raddish peroxidase-conjugatedanti-mouse IgG1 antibody (Bethyl, Montgomery, Tex.) as the detectionantibody. The results indicated a serum half life of 3-5 days. Thelevels of hMCP-1-mIg remained to be about 10 nM at day 7.

Effect of hMCP-1-mIg on acute and relapsing EAE. Experimentalautoimmunine encephalomyelitis (EAE) induction was performed in SJLmice. The mice were immunized on day 0 with 100 μg/mL PLP139-151 peptide(HCLGKWLGHPDKF (SEQ ID NO: 29); Biosynthesis; Lewisville, Tex.) in 2mg/mL complete Freund's adjuvant (CFA) subcutaneously and 100 ng ofpertussis toxin intravenously. Mice were treated on days 3, 5 or on days3, 5, 7 with 10 milligram per kilogram body weight (mpk) of either anmouse IgG1 isotype-control antibody (SP-Biopharma) or the humanMCP-1-mouse Ig (hMCP-1-mIg) fusion protein. Mice were then monitored forbody weight and clinical signs of disease. Disease score was recorded asfollows: 1=limp tail, 2=hind limb weakness, 3=partial hind limbparalysis, 4=total hind limb paralysis, and 5=moribund.

Cell invasion into the central nervous system was monitored by flowcytometry of cells isolated from the spinal cord. An n=3 of mice wereutilized for each group. Mice were perfused with isotonic buffercontaining heparin to remove peripheral blood from the CNS. Brain andspinal cord were harvested for CNS mononuclear isolation. Cells werepushed through a wire mesh, incubated with collagenase and dnase torelease mononuclear cells. The cell suspension was then centrifugedthrough a percoll gradient. Cell recovery was determined by trypan bluecell counts and cellular profile was established through FACS analysis.Cells were stained with BD stains conjugated to antibodies against CD45,CD11b (for inflammatory macrophages), and CD4 (for activated T cells),respectively. The antibodies were purchased from BD Biosciences (SanJose, Calif.). The stained cells were analyzed on Facscaliber using cellquest software (BD Biosciences, San Jose, Calif.).

The results as shown in Table 6 indicated a protecting effect ofhMCP-1-mIg in the EAE model. The protection appeared to be associatedwith the reduction of the number of macrophages in the spinal cord.

TABLE 6 hMCP-1-mlg fusion protein treatment inhibits EAE Maximum MaximumDisease Ave. Ave. Onset Disease Score Onset huMCP-1-Ig Score huMCP-1-IgIgG1 fusion IgG1 fusion Experiment ID Control protein Control proteinExperiment A 11.8 12.0 5, 4, 2 0, 0, 0 Experiment B/C* 10.0 13.5 4, 4, 30, 0, 2 Experiment D 9.6 14.5 4, 4, 4, 4, 3 0, 0, 2, 3, 3 AverageScore-All 10.47 13.33 3.72 0.91 Experiments *Clinical score data wascombined for these two studies. Majority of mice were takenpreclinically for early mechanistic studies and not included.

TABLE 7 hMCP-1-mlg fusion protein inhibits macrophage invasion into theCNS Dosing regime CNS Inflammatory Activated Set 10 mpk analysisTreatment Macrophages* T cells** A d3, 5 d5 hMCP1-mlgG1 1.87E+046.29E+04 mlgG1 2.25E+04 3.76E+04 B d3, 5 d6 hMCP1-mlgG1 1.75E+047.26E+05 mlgG1 4.73E+04 4.12E+05 C d3, 5 d6 hMCP1-mlgG1 1.62E+056.28E+04 mlgG1 3.35E+05 9.20E+04 D d3, 5, 7 d12 hMCP1-mlgG1 0.69E+051.29E+05 mlgG1 2.36E+05 3.72E+05 *Inflammatory Macrophage Populationdefined as CD45hi/CD11b+/CD4− **Activated T cell Population defined asCD45hi/CD11b−/CD4+

Example 6 Effect of hMCP-1-mIg on Anti-Collagen Antibody-InducedArthritis

In this example, the ability of hMCP-1-mIg to reduce anti-collagenantibody-induced arthritis was determined.

Antibody induced arthritis was induced by injecting 800 μg of Chemicon'sarthrogen CIA antibody (Chemicon, Temecula, Calif.) cocktailintravenously into age matched male B10RIII mice. Prior to thisinduction, mice were treated with 40 mpk of either isotype control mIgG1or hMCP-1-mIg fusion protein subcutaneously. Disease onset occurred byday 2 or 3. Animals were scored daily. The scoring system was utilizedto measure each paw as follows: 1=one swollen joint, 2=two or moreswollen joints, 3=entire foot swelling. Each mouse can receive a maximumdisease score of 12. The results obtained are set forth below in Table8.

TABLE 8 MCP-1 Ig fusion protein inhibits antibody induced arthritishuMCP-1 Ig FP in antibody induced arthritis Average Disease Scores Exp 1Exp 2 Exp 1 Exp 2 IgG1 Control huMCP1 Ig FP day 0 0.0 0.0 0.0 0 day 10.0 0.0 0.0 0 day 2 0.5 2.0 0.0 0 day 3 1.5 3.4 0.8 0.2 day 4 3.3 4.00.8 0.6 day 5 3.8 5.6 1.0 0.8 day 6 5.5 5.8 1.3 0.8 day 7 5.3 5.6 1.30.8 day 8 6.0 5.6 1.3 0.8 day 9 5.5 5.4 1.3 0.8 day 10 5.5 5.4 1.3 0.8day 11 5.3 5.0 1.3 0.8 day 12 5.0 4.0 1.3 1.2 day 13 4.5 2.5 1.3 1.2 day14 4.0 1.5 1.3 1.2

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A method for impeding migration of CCR2-bearing cells intoinflammatory tissue, in a subject with atherosclerosis, comprisingadministering, to the subject, a therapeutically effective amount of anisolated polypeptide comprising a human MCPI polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 2 fused to a humanimmunoglobulin gamma-1 polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:
 7. 2. The method of claim 1 wherein thepolypeptide comprising the human MCP1 polypeptide and the humanimmunoglobulin gamma-1 polypeptide comprises one human MCP1 polypeptidefused to one human immunoglobulin gamma-1 polypeptide.
 3. The method ofclaim 2 wherein the polypeptides are fused by a peptide linker.
 4. Themethod of claim 1 wherein the polypeptide comprising the human MCP1polypeptide and the human immunoglobulin gamma-1 polypeptide is in apharmaceutical composition comprising a pharmaceutically acceptablecarrier.
 5. The method of claim 1 wherein the polypeptide comprising thehuman MCP1 polypeptide and the immunoglobulin gamma-1 polypeptide isadministered to the subject in association with one or more furthertherapeutic agents.
 6. The method of claim 5 wherein the furthertherapeutic agent is a member selected from the group consisting ofvaldecoxib; triamcinolone acetonide; tolmetin; tiaprofenic acid;tenoxicam; tazarotene; sulindac; sulfasalazine; salicylic acid;rofecoxib; prednicarbate; piroxicam, salsalate; phenylbutazone;petroleum jelly; penicillamine; oxaprozin; oilated oatmeal; naproxen;nabumetone; mycophenolate mofetil; mometasone furoate; metronidazole;methotrexate; mesalamine; meloxicam; mefenamic acid; meclofenamate;lumiracoxib; ketorolac; ketoprofen; isotretinoin; injectable gold;infliximab; indomethacin; ibuprofen; hydroxyurea; hydroxychloroquine;hydrocortisone; halobetasol propionate; halcinonide; gold sodiumthiomalate; fluticasone propionate; flurbiprofen; flurandrenolide;fluocinonide; fluocinolone acetonide; floctafenine; fenoprofen;etoricoxib; etodolac; etanercept; epsom salts; efalizumab; diflunisal;diflorasone diacetate; diclofenac; desoximetasone; desonide; Dead Seasalts; cyclosporine; coal tar; clobetasol propionate; ciprofloxacin;chloroquine; celecoxib; calcipotriene; budesonide; betamethasonevalerate; betamethasone dipropionate; betamethasone benzoate;azathioprine; aurothioglucose; auranofin; aspirin; anthralin; anakinra;amcinonide; aloe vera; alefacept; alclometasone dipropionate;adalimumab; acitretin; ABX-IL8; a mixture of hydrocortisone,dexamethasone, methylprednisolone and prednisolone; a dietary vitaminB12 supplement; a dietary folate supplement; a dietary calciumsupplement; 6-Thioguanine; and 6-mercaptopurine.
 7. The method of claim1 wherein the polypeptide comprising the human MCP1 polypeptide and thehuman immunoglobulin gamma-1 polypeptide is administered in associationwith a non-steroidal anti-inflammatory drug or a COX-2 inhibitor.